PROJECT SUMMARY Addiction is a chronic relapsing brain disorder that is characterized by compulsive self-administration of abused substances despite their negative consequences. Significant progress has been made in elucidating the molecules and behavioral effects underlying each major drug of abuse. Given that addiction is a disease of interconnected brain networks, examination of network interactions is critical for understanding addiction- related dysfunctions. Resting-state functional connectivity (rsFC), which measures correlations among distinct neurophysiological events in human fMRI studies, has provided insights, but the lack of cellular resolution and inability to carry out experimental perturbation in humans precludes further cause-effect relationship studies. At present, network level analyses are scarce in animal models, resulting in a significant knowledge gap between molecular actions of drugs of abuse and their effects on behavior. In this exploratory application, we aim to uncover how drugs of abuse alter rsFC at systems levels with cellular resolution, through employing brain-wide calcium imaging and computational analyses in larval zebrafish. As a vertebrate genetic model organism, zebrafish shares considerable neuroanatomical and genomic similarity with humans. Larval zebrafish, with a transparent brain of ~100,000 neurons (as compared to ~75 million in the mouse, and ~1 billion in the human brain), is particularly suitable for single-cell resolution analysis of neural circuit activity in vivo. We have established brain-wide calcium imaging and computational data analysis platforms to characterize rsFC at single-cell resolution in larval zebrafish brain. We wish to to test whether single or repeated exposure to morphine will significantly alter rsFC. Impact: If successful, this project will establish a new paradigm to uncover, at systems level and with cellular resolution, how addictive substances alter brain states. Future efforts can expand to all major substances of abuse, which will potentially reveal distinct functional connectivity signatures in acutely or chronically drugged brains. It is also possible to use the platform to examine genetically modified brains and perform small molecule drug screens. We will make technologies including image acquisition, processing, and computational algorithms available to the broad research community. Suitability for CEBRA: (1) This project will develop a new platform to examine the in vivo effects of addictive substances at systems level and with cellular resolution. Such advances are critical to bridge the gap between molecules and behavioral effects of drugs of abuse. At the moment, there are scant precedents or preliminary data regarding this topic, however, if confirmed, it would have a substantial impact on current approaches to understand drugs of abuse. (2) This project will develop innovative technological platforms for neural activity imaging, including image acquisition, processing, and computational algorithms for data analyses. This will have promising applicability to drug abuse research and beyond.